How to develop a successful DUE (drug usage evaluation) program: experience of a teaching medical center.

A drug usage evaluation (DUE) program was successfully implemented in a busy teaching medical center within 12 months of adding a full-time coordinator. The success of this program was due largely to its approval by hospital administration, the recruitment of a pharmacist with the necessary background, and the creation of a structure and process that encouraged participation by leaders of the medical staff. Although the primary objective of hiring the coordinator was to meet Joint Commission on Accreditation of Healthcare Organizations requirements for accreditation, another achievement was confirmed cost savings. Techniques used in selecting DUE subjects and the steps taken in implementing and managing the program are presented.

Expanding formulary and drug-use review programs.

Enlisting the support and participation of physicians is vital if formulary and drug-use review programs are to achieve their full potential for cost savings and quality improvement.

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts.

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts was shown to be stereospecific, activated by glucose starvation and occurred by both high and low affinity systems. Transport by the high affinity system was shown to occur by an active transport process. Furthermore, the high affinity system was shown to be defective in vesicles prepared from F72 cells (hexose transport mutant). These results indicate that the high affinity hexose transport system is retained in the plasma membrane vesicles. Thus plasma membrane vesicles could be of value in further characterization of the L6 high affinity hexose transport system, without interference from the various metabolic events occurring in whole cells.

Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).

Isolation and characterization of hexose transport mutants in L6 rat myoblasts.

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.

Hexose transport in plasma membrane vesicles of rat myoblast L6.

To determine the molecular mechanism of hexose transport in rat myoblasts, transport studies were carried out with purified plasma membrane vesicles. Rat myoblasts were homogenized and fractionated by differential and sucrose gradient centrifugation. Six different fractions were obtained. Studies with marker enzymes revealed that two fractions (A and B) were composed of only plasma membrane. These two fractions differed considerably in their physical properties. Fraction A was composed of large multilaminated vesicles, with an intravesicular volume of 50 microL/mg protein, whereas fraction B was composed of membrane fragments and much smaller vesicles, with an intravesicular volume of 7 microL/mg protein. Based on the response of the ouabain-sensitive Na+, K+-ATPase activity to sodium dodecyl sulfate and ionophore treatments, it seemed likely that fraction A was composed of a significant amount of sealed right-side-out vesicles, whereas fraction B was composed of mainly membrane sheets or leaky vesicles. The initial rate of hexose influx into the membrane vesicles was determined by the flow dialysis technique. The optimal conditions for 2-deoxyglucose (2-DG) uptake into the plasma membrane vesicles were either 50 mM phosphate buffer or 10 mM 2-(N-2-hydroxyethylpiperazin-N'-yl)ethanesulfonic acid buffer at pH 7.0. In the presence of 500 microM 2-DG, the initial rates of 2-DG influx were 295 and 49 nmol/min per milligram protein for fractions A and B, respectively. In other words, after 1 min of incubation, the intravesicular concentration of 2-DG was around 6 mM, about 10 times the extravesicular concentration. D-Glucose was taken up to a similar extent (333 nmol/min per milligram protein), whereas L-glucose only equilibrated across the plasma membrane. Analysis of the fate of 2-DG revealed that the substrate was not phosphorylated upon incubation with the vesicles. Transport activity can be abolished either by disruption of the membrane vesicles or by reduction of the electrical potential across the membrane.

Cardiovascular malformations associated with administration of prenalterol to young chick embryos.

Prenalterol (levo-1-[4-hydroxyphenoxy]-3-isopropyl-amino-2-propanol), a new stimulant of cardiac beta-adrenergic receptors in man, induces cardiovascular malformations when topically administered to 2 1/2- through 5 1/2-day chick embryos (Hamburger-Hamilton stages 17-27). Ventricular septal defects (VSD) located in the middle portion of the conal septum and classified as the simple, punched-out type VSD without septal malalignment were the predominant malformations observed throughout the developmental period tested. Arch malformations of the aortic circulation were also observed throughout the test interval, while anomalies of the pulmonary system were observed only at Hamburger-Hamilton stages 17 and 26-27. At stage 25 prenalterol demonstrated an acute toxicity significantly less than (1/50-1/20) epinephrine (P = .035 at concentrations of 8-10 mM) but was relatively equipotent with epinephrine in producing cardiovascular malformations. The effective median concentrations of the two agents were comparable (0.5-1.0 mM). The spectra of malformations induced by prenalterol and epinephrine were qualitatively similar. Malformations included absence of the right and/or left third aortic arch (innominate arteries), persistent remnant of the left fourth aortic arch, and VSD. These results support a previously proposed theory by these investigators that hyperstimulation of cardiac beta-adrenergic receptors in the chick embryo produces cardiovascular malformations.

Acetylsalicylic acid-induced morphological changes in the ductus arteriosus of the chick embryo.

The effect of acetylsalicyclic acid upon the ducti arteriosi of the embryonic chick was studied. A spectrum of gross malformations and histological findings associated with premature closure of the right ductus arteriosus is presented.

Aortic aneurysm associated with cardiac defects in theophyline stimulated chick embryos.

Theophylline(1 ml 0.02 M in 0.15 M saline) was administered to embryonic chicks (Hamburger-Hamilton developmental stages 24.27). The drug was topically applied to the surface of the chorioallantoic membrane in the vicinity of the embryonic heart. At necropsy, 63% (44/70) of the theophylline-exposed embryos demonstrated an aneurysm in the ascending aorta. Among the cases exhibiting aortic aneurysm, 48% (21/44) of the embryos demonstrated ventricular septal defects. Four cases of truncus arteriosus were also observed. The morphogenesis and mechanism of theophylline-induced cardiac defects with accompanying aortic aneurysm may be related to the development of the conotruncal region of the heart.

Chronotropism and blood flow patterns following teratogenic doses of catecholamines in 5-day-old chick embryos.

In vivo heart rates of 5-day-old chick embryos were recorded from electrodes placed in close proximity to the heart. L-epinephrine (4X10(-10) mole), 1-norepinephrine (1X10(-9)mole) and 1-isoproterenol (1.6X10(-10)mole) in 5 microliter of isotonic saline transiently accerlerated the mean heart rate by almost 9 percent. L-phenylephrine (2X10(-9)mole/5microliter) and the experimental procedure produced no appreciable effect. The positive chronotropic effect of the catecholamines was found to be highly significant (P less than 0.0005) as computed by Student's t test. However, no direct relationship could be established between the chronotropic response and the aortic arch anomalies produced. A prolonged reduction of blood flow in the primitive heart tube and the sixth aortic arch after administration of epinephrine and isoproterenol is apparently related to the induction of hypoplastic right pulmonary artery with absent or hypoplastic right ductus arteriosus.

The effects of methylxanthines on catecholamine-stimulated and normal chick embryos.

Dose of theophylline and caffeine which do not produce aortic arch anomalies in embryonic chicks have been shown to potentiate catecholamine-induced aortic arch malformations in that experimental animal. Theophylline (2.1 X 10(-5) mole per milliliter isotonic saline solution) potentiated the effective dose of norepinephrine more than 100 times. The greatest potentiation observed with epinephrine (2.5 X) was induced by 2.6 X 10(-5) mole caffeine. This study also demonstrated that both methylxanthines specifically induce aneurysms of the ascending aorta and complete absence (or nearly complete constriction) of the right ductus arteriosus. The incidences of these types of cardiovascular malformations proved to be dose dependent with theophylline a more potent teratogen than caffeine. The mobilization of calcium and/or cyclic nucleotide phosphodiesterase inhibition by the methylxanthines are suggested as significant actions in the potentiation of catecholamine-induced aortic arch anomalies.

The effect of practolol and butoxamine on aortic arch malformation in beta adrenoreceptor stimulated chick embryos.

An equimolar dose of the beta-1 adrenoreceptor antagonist practolol administered to embryonic chicks prevents the induction of aortic arch malformations by isoproterenol. Whereas 3.75 X 10(-9) mole isoproterenol in 5 microliter saline solution induced aortic arch anomalies in 39% of embryos injected at Hamburger-Hamilton developmental stage 26, pretreatment with practolol one to two minutes before catecholamine administration reduced the anomaly rate to to 4%. Practolol when injected alone did not influence survival rate nor did it cause cardiovascular malformations. Probably the most significant result of this study involves the prevention by practolol of aortic hypoplasia and interrupted aortic arch complexes, anomalies frequently induced by isoproterenol when administered at this stage of embryonic chick development. Butoxamine, a beta-2 adrenoreceptor antagonist, did not block the overall effect of isoproterenol nearly as effectively as did practolol. Results from the present study suggest that aortic arch anomalies may be induced in embryonic chicks via beta-1 adrenoreceptor stimulation. Beta-2 receptor stimulation does not appear to be as significantly involved.